Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species.

Akhikun Nahar,David S Nichols,Anthony L Baker,Margaret L Britz,John P Bowman

doi:10.3390/ijms21051670

Akhikun Nahar, David S Nichols + Show 3 more

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https://doi.org/10.3390/ijms21051670

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Abstract

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

Highlights

Lipids are important cellular components of biological systems and are involved in membrane formation of cells and cellular structures, signaling and energy storage [1]
To confirm the presence of mycolic acids (MA), TAG and FFA in bacterial extracts, lipid extracts of six sub-Antarctic strains and the Corynebacterium glutamicum control were prepared by bead beating freeze-dried cells in chloroform:methanol (2:1, v/v), the extracted lipids and commercial standards were separated on silica-TLC plates
The nature of these lipids, the major lipid detected at the solvent front, was further evaluated by thin-layer chromatography-flame ionization detection (TLC-flame-ionization detector (FID)) and other analyses, as described below

Summary

Introduction

Lipids are important cellular components of biological systems and are involved in membrane formation of cells and cellular structures, signaling and energy storage [1]. Membrane lipids are mainly phospholipids (PL) in the form of two fatty acid (FA) chains linked to glycerol–phosphate derivatives, where the structures vary amongst bacterial species. Along with cell wall MA, some species of this group can accumulate triacylglycerols (TAG) as storage lipids, which are considered as potential substrates for biofuel development [6]. The most studied lipid-accumulating genus is Rhodococcus, which contains many species considered to have industrial potential for TAG synthesis [7,8,9,10,11]. Quantitative monitoring of the distribution of carbon into the suite of storage and structural lipids (PL, MA and other surface lipid structures) is hampered by the lack of a single method to quantify all of the lipids

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